Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
     
       
         
         
             
             
         
       
     
     where R 1 , R 2 , R 3 , X, R 4 , R 5 , and R 6  are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/491,749, filed on May 31, 2011; the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to novel compounds havingneprilysin-inhibition activity. The invention also relates topharmaceutical compositions comprising such compounds, processes andintermediates for preparing such compounds and methods of using suchcompounds to treat diseases such as hypertension, heart failure,pulmonary hypertension, and renal disease.

State of the Art

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many organsand tissues, including the brain, kidneys, lungs, gastrointestinaltract, heart, and the peripheral vasculature. NEP degrades andinactivates a number of endogenous peptides, such as enkephalins,circulating bradykinin, angiotensin peptides, and natriuretic peptides,the latter of which have several effects including, for example,vasodilation and natriuresis/diuresis, as well as inhibition of cardiachypertrophy and ventricular fibrosis. Thus, NEP plays an important rolein blood pressure homeostasis and cardiovascular health.

NEP inhibitors, such as thiorphan, candoxatril, and candoxatrilat, havebeen studied as potential therapeutics. Compounds that inhibit both NEPand angiotensin-I converting enzyme (ACE) are also known, and includeomapatrilat, gempatrilat, and sampatrilat. Referred to as vasopeptidaseinhibitors, this latter class of compounds is described in Robl et al.(1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess neprilysin (NEP) enzyme inhibition activity. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating conditions such as hypertension andheart failure.

One aspect of the invention relates to a compound of formula I:

where:

R¹ is —OR¹⁰ or —NR⁶⁰R⁷⁰;

R² is H or —OR²⁰;

R³ is selected from H, Cl, F, —CH₃, and —CF₃;

X is a —C₁₋₁₀heteroaryl or a partially unsaturated —C₂₋₁₂heterocycle,with the proviso that X is not pyrazole;

R⁴ is absent or is selected from H; halo; —C₀₋₅alkylene-OH; —NH₂;—C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl; —C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R⁴⁰;—C₀₋₁alkylene-C(O)OR⁴¹; —C(O)NR⁴²R⁴³; —NHC(O)R⁴⁴; ═O; —NO₂; furan;pyrazine; naphthalene; pyridine; pyrazole optionally substituted withmethyl; thiophene optionally substituted with methyl; and phenyloptionally substituted with one group selected from halo, —OH, —CF₃,—OCH₃, —NHC(O)CH₃, and phenyl; and R⁴, when present, is attached to acarbon atom; and R⁴ can also be phenyl substituted with —C(O)OR⁴¹ when Xis pyridazine, pyrazine, or pyridine;

R⁵ is absent or is selected from H; —C₁₋₆alkyl; —C₀₋₃alkylene-OH;—[(CH₂)₂O]₁₋₃CH₃; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²;—C₀₋₂alkylene-pyridine optionally substituted with halo; —CH₂-isoxazoleoptionally substituted with methyl; —CH₂-pyrimidine optionallysubstituted with —O—C₁₋₆alkyl; —C₂alkylene-phenyl;

and R⁵, when present, is attached to a nitrogen atom;

R⁶ is absent or is selected from H, halo, —OH, —C₁₋₆alkyl, and—O—C₁₋₆alkyl; and R⁶, when present, is attached to a carbon atom;

R¹⁰, R⁴¹ and R⁵⁰ are independently selected from H, —C₁₋₆alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵,—C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃;R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl,or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or—(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R¹⁷ isselected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵;

R²⁰ is H or is taken together with R¹⁰ to form —CR²¹R²²— or is takentogether with R⁶⁰ to form —C(O)—; where R²¹ and R²² are independentlyselected from H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R²¹ and R²² aretaken together to form ═O;

R⁴⁰ is H or —C₁₋₆alkyl;

R⁴², R⁴³, R⁵¹, and R⁵² are independently selected from H, —C₁₋₆alkyl,—CH₂COOH, —(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂,—C₃₋₇cycloalkyl, and —(CH₂)₂-imidazole; or R⁴² and R⁴³ or R⁵² and R⁵³aretaken together to form a saturated or partially unsaturated—C₃₋₅heterocycle optionally substituted with —OH, —COOR⁴¹, or —CONH₂,and optionally containing an oxygen atom in the ring;

R⁴⁴ is selected from —C₁₋₆alkyl; —C₀₋₁alkylene-O—C₁₋₆alkyl; phenyloptionally substituted with halo or —OCH₃; and —C₁₋₉heteroaryl; and

R⁵³, R⁵⁴, R⁵⁵, R⁵⁶, and R⁵⁷ are independently selected from H, halo,—C₁₋₆alkyl, —O—C₁₋₆alkyl, and —S—C₁₋₆alkyl, where each —C₁₋₆alkyl isoptionally substituted with 1-5 fluoro atoms;

R⁶⁰ is selected from H, —OH, —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl,and —OC(S)NR⁶²R⁶³; where R⁶¹ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl,—OCH₂—C₆₋₁₀aryl, —CH₂O—C₆₋₁₀aryl, and —NR⁶²R⁶³; R⁶² and R⁶³ areindependently H or —C₁₋₄alkyl;

R⁷⁰ is selected from H, —C₁₋₆alkyl, and —C(O)R⁷¹; R⁷¹ is selected fromH, —C₁₋₆alkyl, —C₃₋₇cycloalkyl, —C₆₋₁₀aryl, and —C₁₋₉heteroaryl;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention as thefirst therapeutic agent, one or more secondary therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess NEP enzyme inhibition activity, andare therefore expected to be useful as therapeutic agents for treatingpatients suffering from a disease or disorder that is treated byinhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension,heart failure, or renal disease, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Stillanother aspect of the invention relates to a method for inhibiting a NEPenzyme in a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Since compounds of the invention possess NEP inhibition activity, theyare also useful as research tools. Accordingly, one aspect of theinvention relates to a method of using a compound of the invention as aresearch tool, the method comprising conducting a biological assay usinga compound of the invention. Compounds of the invention can also be usedto evaluate new chemical compounds. Thus another aspect of the inventionrelates to a method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. Still another aspect of the invention relates to amethod of studying a biological system or sample comprising a NEPenzyme, the method comprising: (a) contacting the biological system orsample with a compound of the invention; and (b) determining the effectscaused by the compound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of formula I, comprising the steps of: (a) couplingcompound 1 with compound 2:

or(b) coupling compound 1 with compound 2a to form compound 3:

where L is a leaving group such as a halogen (e.g., bromo) or a triflate(e.g., —OSO₂CF₃), and reacting compound 3 with optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane:

in a palladium-catalyzed coupling reaction; or (c) reacting compound 2bwith optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4 ina palladium-catalyzed coupling reaction:

where L is a leaving group and P¹ is a carboxy-protecting group selectedfrom methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl,trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl, and couplingcompound 4 with compound 1; and (d) optionally deprotecting the productof step (a) or (b) or (c), to produce a compound of formula I or apharmaceutically acceptable salt thereof; where R¹, R², R³, X, R⁴, R⁵,and R⁶ are as defined for formula I. Another aspect of the inventionrelates to a process of preparing a pharmaceutically acceptable salt ofa compound of formula I, comprising contacting a compound of formula Iin free acid or base form with a pharmaceutically acceptable base oracid. In other aspects, the invention relates to products prepared byany of the processes described herein, as well as novel intermediatesused in such process. In one aspect of the invention novel intermediateshave formula II, as defined herein.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal. Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to carbon atoms and include, for example,—C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, and —C₁₋₁₀alkyl.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₆alkylene-, and —C₁₋₃alkylene-.Representative alkylene groups include, by way of example, methylene,ethane-1,2-diyl (“ethylene”), propane-1,2-diyl, propane-1,3-diyl,butane-1,4-diyl, pentane-1,5-diyl and the like. It is understood thatwhen the alkylene term include zero carbons such as —C₀₋₁alkylene-, suchterms are intended to include the absence of carbon atoms, that is, thealkylene group is not present except for a covalent bond attaching thegroups separated by the alkylene term.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or one or more fused rings. Fused ring systemsinclude those that are fully unsaturated (e.g., naphthalene) as well asthose that are partially unsaturated (e.g.,1,2,3,4-tetrahydronaphthalene). Unless otherwise defined, such arylgroups typically contain from 6 to 10 carbon ring atoms and include, forexample, —C₆₋₁₀aryl. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heterocycle” is intended to include monovalent unsaturated(aromatic) heterocycles having a single ring or two fused rings as wellas monovalent saturated and partially unsaturated groups having a singlering or multiple condensed rings. The heterocycle ring can contain from3 to 15 total ring atoms, of which 1 to 14 are ring carbon atoms, and 1to 4 are ring heteroatoms selected from nitrogen, oxygen or sulfur.Typically, however, the heterocycle ring contains from 3 to 10 totalring atoms, of which 1 to 9 are ring carbon atoms, and 1 to 4 are ringheteroatoms. The point of attachment is at any available carbon ornitrogen ring atom. Exemplary heterocycles include, for example,—C₁₋₇heterocycle, —C₃₋₅heterocycle, —C₂₋₆heterocycle, —C₃₋₁₂heterocycle,—C₅₋₉heterocycle, —C₁₋₉heterocycle, —C₁₋₁₁heterocycle, and—C₁₋₁₄heterocyle.

Monovalent unsaturated heterocycles are also commonly referred to as“heteroaryl” groups. Unless otherwise defined, heteroaryl groupstypically contain from 5 to 10 total ring atoms, of which 1 to 9 arering carbon atoms, and 1 to 4 are ring heteroatoms, and include, forexample, —C₁₋₉heteroaryl and —C₅₋₉heteroaryl. Representative heteroarylgroups include, by way of example, pyrrole (e.g., 3-pyrrolyl and2H-pyrrol-3-yl), imidazole (e.g., 2-imidazolyl), furan (e.g., 2-furyland 3-furyl), thiophene (e.g., 2-thienyl), triazole (e.g.,1,2,3-triazolyl and 1,2,4-triazolyl), pyrazole (e.g., 1H-pyrazol-3-yl),oxazole (e.g., 2-oxazolyl), isoxazole (e.g., 3-isoxazolyl), thiazole(e.g., 2-thiazolyl and 4-thiazolyl), and isothiazole (e.g.,3-isothiazolyl), pyridine (e.g., 2-pyridyl, 3-pyridyl, and 4-pyridyl),pyridylimidazole, pyridyltriazole, pyrazine, pyridazine (e.g.,3-pyridazinyl), pyrimidine (e.g., 2-pyrimidinyl), tetrazole, triazine(e.g., 1,3,5-triazinyl), indolyle (e.g., 1H-indol-2-yl, 1H-indol-4-yland 1H-indol-5-yl), benzofuran (e.g., benzofuran-5-yl), benzothiophene(e.g., benzo[b]thien-2-yl and benzo[b]thien-5-yl), benzimidazole,benzoxazole, benzothiazole, benzotriazole, quinoline (e.g., 2-quinolyl),isoquinoline, quinazoline, quinoxaline and the like.

Monovalent saturated heterocycles typically contain from 3 to 10 totalring atoms, of which 2 to 9 are ring carbon atoms, and 1 to 4 are ringheteroatoms, and include, for example —C₃₋₅heterocycle. Representativemonovalent saturated heterocycles include, by way of example, monovalentspecies of pyrrolidine, imidazolidine, pyrazolidine, piperidine,1,4-dioxane, morpholine, thiomorpholine, piperazine, 3-pyrroline and thelike. In some instances, moieties may be described as being takentogether to form a saturated —C₃₋₅heterocycle optionally containing anoxygen atom in the ring. Such groups include:

Monovalent partially unsaturated heterocycles typically contain from 3to 10 total ring atoms, of which 2 to 11 are ring carbon atoms, and 1 to3 are ring heteroatoms, and include, for example —C₃₋₅heterocycle and—C₂₋₁₂heterocycle. Representative monovalent partially unsaturatedheterocycles include, by way of example, pyran, benzopyran, benzodioxole(e.g., benzo[1,3]dioxol-5-yl), tetrahydropyridazine,2,5-dihydro-1H-pyrrole, dihydroimidazole, dihydrotriazole,dihydrooxazole, dihydroisoxazole, dihydrothiazole, dihydroisothiazole,dihydrooxadiazole, dihydrothiadiazole, tetrahydropyridazine,hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene. In someinstances, moieties may be described as being taken together to form apartially unsaturated —C₃₋₅heterocycle. Such groups include:

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times or 1 to 5 times. For example, a phenyl group that is“optionally substituted” with halo atoms, may be unsubstituted, or itmay contain 1, 2, 3, 4, or 5 halo atoms.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. The term is also intended to includecertain protected derivatives of compounds of formula I that may be madeprior to a final deprotection stage. Such compounds may not possesspharmacological activity at NEP, but may be administered orally orparenterally and thereafter metabolized in the body to form compounds ofthe invention which are pharmacologically active at NEP. Thus, allprotected derivatives and prodrugs of compounds formula I are includedwithin the scope of the invention. Prodrugs of compounds of formula Ihaving a free carboxyl group can be readily synthesized by techniquesthat are well known in the art. These prodrug derivatives are thenconverted by solvolysis or under physiological conditions to be the freecarboxyl compound. Exemplary prodrugs include esters such asC₁₋₆alkylesters and aryl-C₁₋₆alkylesters. In one embodiment, thecompounds of formula I have a free carboxyl and the prodrug is an esterderivative thereof, i.e., the prodrug is an ester such as —C(O)OCH₂CH₃.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in anassay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas Ia-Il, as well as the compounds encompassed by formula II. Inaddition, the compounds of the invention may also contain several basicor acidic groups (for example, amino or carboxyl groups) and therefore,such compounds can exist as a free base, free acid, or in various saltforms. All such salt forms are included within the scope of theinvention. Furthermore, the compounds of the invention may also exist asprodrugs. Accordingly, those skilled in the art will recognize thatreference to a compound herein, for example, reference to a “compound ofthe invention” or a “compound of formula I” includes a compound offormula I as well as pharmaceutically acceptable salts and prodrugs ofthat compound unless otherwise indicated. Further, the term “or apharmaceutically acceptable salt and/or prodrug thereof” is intended toinclude all permutations of salts and prodrugs, such as apharmaceutically acceptable salt of a prodrug. Furthermore, solvates ofcompounds of formula I are included within the scope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention also relates to racemicmixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the terms “compound offormula I,” “compounds of formula II,” and so forth, are intended toinclude all possible stereoisomers of the compound. Similarly, when aparticular stereoisomer is shown or named herein, it will be understoodby those skilled in the art that minor amounts of other stereoisomersmay be present in the compositions of the invention unless otherwiseindicated, provided that the utility of the composition as a whole isnot eliminated by the presence of such other isomers. Individualstereoisomers may be obtained by numerous methods that are well known inthe art, including chiral chromatography using a suitable chiralstationary phase or support, or by chemically converting them intodiastereoisomers, separating the diastereoisomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified. For example, if X is depicted as (R⁵ beinghydrogen):

it is understood that the compound may also exist in a tautomeric formsuch as:

More specifically, compounds of formula I contain at least one chiralcenter when R² is H, and contain at least two chiral centers when R² is—OR²⁰. These chiral centers are indicated by the symbols * and ** in thefollowing formulas Ia and Ib:

In one stereoisomer of the compounds of formula Ia, the carbon atomidentified by the ** symbol has the (R) configuration. This embodimentof the invention is shown in formula Ia-1:

In this embodiment, compounds have the (R) configuration at the **carbon atom or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another stereoisomer of thecompounds of formula Ia, the carbon atom identified by the ** symbol hasthe (S) configuration. This embodiments of the invention is shown informula Ia-2:

In this embodiment, compounds have the (S) configuration at the **carbon atom or are enriched in a stereoisomeric form having the (S)configuration at this carbon atom.

In one stereoisomer of the compound of formula Ib, both carbon atomsidentified by the * and ** symbols have the (R) configuration. Thisembodiment of the invention is shown in formula Ib-1:

In this embodiment, compounds have the (R,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,R) configuration at these carbon atoms. In another stereoisomer ofthe compound of formula Ib, both carbon atoms identified by the * and **symbols have the (S) configuration. This embodiment of the invention isshown in formula Ib-2:

In this embodiment, compounds have the (S,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,S) configuration at these carbon atoms. In yet another stereoisomerof the compound of formula Ib, the carbon atom identified by thesymbol * has the (S) configuration and the carbon atom identified by thesymbol ** has the (R) configuration. This embodiment of the invention isshown in formula Ib-3:

In this embodiment, compounds have the (S,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,R) configuration at these carbon atoms. In still another stereoisomerof the compound of formula Ib, the carbon atom identified by thesymbol * has the (R) configuration and the carbon atom identified by thesymbol ** has the (S) configuration. This embodiment of the invention isshown in formula Ib-4:

In this embodiment, compounds have the (R,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,S) configuration at these carbon atoms.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of formulaI enriched in deuterium especially at a site of metabolism resulting,for example, in compounds having greater metabolic stability; andcompounds of formula I enriched in a positron emitting isotope, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one embodiment, this invention relates to compounds of formula I:

R¹ is selected from —OR¹⁰ and —NR⁶⁰R⁷⁰. The R¹⁰ moiety is selected fromH, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³,—C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

where R¹³ is selected from —C₁₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃;R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl,or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or—(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁶⁰ moietyis selected from H, —OH, —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and—OC(S)NR⁶²R⁶³; where R⁶¹ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl,—OCH₂—C₆₋₁₀aryl, —CH₂O—C₆₋₁₀aryl, and —NR⁶²R⁶³; and R⁶² and R⁶³ areindependently H or —C₁₋₄alkyl. The R⁷⁰ moiety is selected from H,—C₁₋₆alkyl, and —C(O)R⁷¹; where R⁷¹ is selected from H, —C₁₋₆alkyl,—C₃₋₇cycloalkyl, —C₆₋₁₀aryl, and —C₁₋₉heteroaryl.

In one embodiment, R¹ is —OR¹⁰, and R¹⁰ is H or —C₁₋₆alkyl (e.g.,—CH₂CH₃ and —(CH₂)₃CH₃).

In one embodiment, R¹ is selected from —OR¹⁰ and —NR⁶⁰R⁷⁰, where R¹⁰ isH, R⁶⁰ is H or —OH, and R⁷⁰ is H.

In another embodiment, R¹ is —OR¹⁰, where R¹⁰ is selected from—C₁₋₆alkyl (e.g., —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂,—(CH₂)₃CH₃, —(CH₂)₄CH₃, and —(CH₂)₂CH(CH₃)₂), —C₁₋₃alkylene-C₆₋₁₀aryl(e.g., benzyl), —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃ (e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃),—C₁₋₆alkylene-OC(O)R¹³ (e.g., —CH₂—OC(O)CH₃, —CH₂—OC(O)CH₂CH₃, and—CH₂—OC(O)OCH₂CH₃), —C₁₋₆alkylene-NR¹⁴R¹⁵ (e.g., —(CH₂)₂—N(CH₃)₂,

—C₁₋₆alkylene-C(O)R¹⁷ (e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl,—CH₂C(O)—N(CH₃)₂, and

—C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl (e.g.,—(CH₂)₂SO₂CH₃),

In another embodiment, R¹ is —NR⁶⁰R⁷⁰, where R⁶⁰ is selected from—OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ isH. In still another embodiment, R¹ is —NR⁶⁰R⁷⁰, where R⁶⁰ is H or —OH,and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹. In yet another embodiment, R¹ is—NR⁶⁰R⁷⁰, where R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl,pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹. In oneaspect of the invention, these compounds may find particular utility asprodrugs or as intermediates in the synthetic procedures describedherein. For example, in one embodiment, R¹ is —OR¹⁰ and R¹⁰ is—C₁₋₆alkylene-OC(O)R¹³, such as —O—CH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R¹ is —OR¹⁰ and R¹⁰ is—C₀₋₆alkylenemorpholine such as —O—(CH₂)₂-morpholine:

making the compound a 2-morpholinoethyl or mofetil ester; or R¹ is —OR¹⁰and R¹⁰ is

such as —O—CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

R² is selected from H and —OR²⁰. The R²⁰ moiety is H or is takentogether with R¹⁰ to form —CR²¹R²²— or is taken together with R⁶⁰ toform —C(O)—; where R²¹ and R²² are independently selected from H,—C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R²¹ and R²² are taken together toform ═O. In one embodiment, R² is selected from H and —OR²⁰, where R²⁰is H.

When R² is —OR²⁰ and R²⁰ is taken together with R¹⁰ to form —CR²¹R²²—,this embodiment can be depicted as:

and when R²¹ and R²² are taken together to form ═O, this embodiment canbe depicted as:

When R² is —OR²⁰ and R²⁰ is taken together with R⁶⁰ to form —NHC(O)—,this embodiment can be depicted as:

In one aspect of the invention, these compounds may find particularutility as prodrugs or as intermediates in the synthetic proceduresdescribed herein.

R³ is selected from H, Cl, F, —CH₃, and —CF₃. In one embodiment, R³ isselected from H, Cl, and —CF₃.

The “X” moiety is a —C₁₋₁₀heteroaryl or a partially unsaturated—C₃₋₁₂heterocycle, with the proviso that X is not pyrazole. Compoundswhere X is a pyrazole are described in commonly-assigned U.S.Provisional Application No. 61/491,750 filed on May 31, 2011 and U.S.application Ser. No. ______ filed on even date herewith (Attorney DocketNo. P-285-US1), both entitled “Neprilysin Inhibitors” to Gendron et al.Note that in some embodiments, R⁴, R⁵, and/or R⁶ may be absent. Whenpresent, R⁴ and/or R⁶ are on any available carbon atom. When present, R⁵is on any available nitrogen atom. In one embodiment, X″ moiety is a—C₁₋₁₀heteroaryl, examples of which include:

pyrrole:

furan:

thiophene:

imidazole such as:

specific examples of which include:

triazole, including 1,2,3-triazole such as:

as well as 1,2,4-triazole such as:

oxazole:

specific examples of which include:

isoxazole:

specific examples of which include:

thiazole:

specific examples of which include:

isothiazole:

oxadiazole, including [1,2,4]oxadiazole such as:

as well as [1,2,3]oxadiazole such as:

and [1,3,4]oxadiazole:

thiadiazole, including [1,2,4]thiadiazole such as:

as well as [1,2,3]thiadiazole such as:

and [1,3,4]thiadiazole:

tetrazole such as:

pyridine:

specific examples of which include:

pyrazine:

pyrimidine:

specific examples of which include:

pyridazine:

benzimidazole such as:

specific examples of which include:

benzotriazole:

specific examples of which include:

benzoxazole such as:

specific examples of which include:

benzothiazole such as:

specific examples of which include:

pyridylimidazole such as:

a specific example of which includes:

pyridyltriazole such as:

specific examples of which include:

imidazopyridine such as:

a specific example of which includes:

pyrrolopyrimidine such as:

a specific example of which includes:

and

5-oxa-3,3a-diazacyclopenta[a]naphthalene:

In one embodiment, X is a partially unsaturated —C₂₋₁₂heterocycle,examples of which include:

dihydroimidazole:

dihydrotriazole such as:

dihydrooxazole:

dihydroisoxazole:

dihydrothiazole:

dihydroisothiazole:

dihydrooxadiazole

dihydrothiadiazole:

tetrahydropyridazine:

hexahydropyrroloquinoxaline such as

a specific example of which includes:

and

dihydrooxadiazabenzo[e]azulene:

In one particular embodiment, X is selected from pyrrole, furan,thiophene, imidazole, triazole, oxazole, isoxazole, thiazole,isothiazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyrazine,pyrimidine, pyridazine, benzimidazole, benzotriazole, benzoxazole,benzothiazole, pyridylimidazole, pyridyltriazole, imidazopyridine,pyrrolopyrimidine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene,dihydroimidazole, dihydrotriazole, dihydrooxazole, dihydroisoxazole,dihydrothiazole, dihydroisothiazole, dihydrooxadiazole,dihydrothiadiazole, tetrahydropyridazine, hexahydropyrroloquinoxaline,and dihydrooxadiazabenzo[e]azulene.

In one particular embodiment, X is selected from furan, thiophene,imidazole, triazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, benzimidazole, benzotriazole, pyridylimidazole,pyridyltriazole, imidazopyridine, pyrrolopyrimidine,5-oxa-3,3a-diazacyclopenta[a]naphthalene, dihydrotriazole,dihydroisoxazole, tetrahydropyridazine, hexahydropyrroloquinoxaline, anddihydrooxadiazabenzo[e]azulene.

The R⁴ moiety may be absent. When present, the R⁴ moiety is attached toa carbon atom and is selected from H; halo; —C₀₋₅alkylene-OH; —NH₂;—C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl; —C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R⁴⁰;—C₀₋₁alkylene-C(O)OR⁴¹; —C(O)NR⁴²R⁴³; —NHC(O)R⁴⁴; ═O; —NO₂; furan;pyrazine; naphthalene; pyridine; pyrazole optionally substituted withmethyl; thiophene optionally substituted with methyl; and phenyloptionally substituted with one group selected from halo, —OH, —CF₃,—OCH₃, —NHC(O)CH₃, and phenyl; and R⁴ can also be phenyl substitutedwith —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine. The R⁴⁰moiety is H or —C₁₋₆alkyl. The R⁴¹ moiety is selected from H,—C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³,—C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃;R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl,or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or—(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁴² and R⁴³moieties are independently selected from H, —C₁₋₆alkyl, —CH₂COOH,—(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂, —C₃₋₇cycloalkyl,and —(CH₂)₂-imidazole; or R⁴² and R⁴³ are taken together to form asaturated or partially unsaturated —C₃₋₅heterocycle optionallysubstituted with —OH, —COOR⁴¹, or —CONH₂, and optionally containing anoxygen atom in the ring. The R⁴⁴ moiety is selected from —C₁₋₆alkyl;—C₀₋₁alkylene-O—C₁₋₆alkyl; phenyl optionally substituted with halo or—OCH₃; and —C₁₋₉heteroaryl.

In one embodiment, R⁴ is selected from H; halo (e.g. chloro, fluoro, andbromo); —C₀₋₅alkylene-OH (e.g., —OH); —C₁₋₆alkyl (e.g., —CH₃, —CH₂CH₃,—(CH₂)₂CH₃, and —(CH₂)₃CH₃); —C₀₋₁alkylene-C(O)OR⁴¹ (e.g., —COOR⁴¹); ═O;phenyl optionally substituted with one halo (e.g., chloro); and phenylsubstituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine;and R⁴¹ is selected from H and —C₁₋₆alkyl (e.g., —CH₂CH₃).

In one particular embodiment, R⁴ is —C₀₋₁alkylene-C(O)OR⁴¹, where R⁴¹ isH. In another particular embodiment, R⁴ is —C₀₋₁alkylene-C(O)OR⁴¹, whereR⁴¹ is selected from —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷,—C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these latter compounds may findparticular utility as prodrugs or as intermediates in the syntheticprocedures described herein.

The R⁵ moiety may be absent. When present, the R⁵ moiety is attached toa nitrogen atom and is selected from H; —C₁₋₆alkyl; —C₀₋₃alkylene-OH;—[(CH₂)₂O]₁₋₃CH₃; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²;—C₀₋₂alkylene-pyridine optionally substituted with halo; —CH₂-isoxazoleoptionally substituted with methyl; —CH₂-pyrimidine optionallysubstituted with —O—C₁₋₆ alkyl; —C₂alkylene-phenyl;

The R⁵⁰ moiety is selected from H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷,—C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

where R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃;R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl,or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or—(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁵¹ and R⁵²moieties are independently selected from H, —C₁₋₆alkyl, —CH₂COOH,—(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂, —C₃₋₇cycloalkyl,and —(CH₂)₂-imidazole; or R⁵² and R⁵³are taken together to form asaturated or partially unsaturated —C₃₋₅heterocycle optionallysubstituted with —OH, —COOR⁴¹, or —CONH₂, and optionally containing anoxygen atom in the ring. The R⁵³, R⁵⁴, R⁵⁵, R⁵⁶, and R⁵⁷ moieties areindependently selected from H, halo, —C₁₋₆alkyl, —O—C₁₋₆alkyl, and—S—C₁₋₆alkyl, where each —C₁₋₆alkyl is optionally substituted with 1-5fluoro atoms.

In one embodiment, R⁵ is absent or is selected from H; —C₀₋₃alkylene-OH(e.g., —OH and —(CH₂)₃OH); —C₁₋₃alkylene-C(O)OR⁵⁰ (e.g., —CH₂C(O)OR⁵⁰);—CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted withhalo (e.g., bromo), for example:

where R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is selected from H, halo (e.g.,fluoro), and —O—C₁₋₆alkyl (e.g., —OCH₃); R⁵⁴ is selected from H and halo(e.g., fluoro); R⁵⁵ is selected from H, halo (e.g., chloro), and—O—C₁₋₆alkyl (e.g., —OCH₃); R⁵⁶ is H; and R⁵⁷ is H.

In one particular embodiment, R⁵ is —C₁₋₃alkylene-C(O)OR⁵⁰, where R⁵⁰ isH. In another particular embodiment, R⁵ is —C₁₋₃alkylene-C(O)OR⁵⁰, whereR⁵⁰ is selected from —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷,—C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these latter compounds may findparticular utility as prodrugs or as intermediates in the syntheticprocedures described herein.

The R⁶ may be absent. When present, the R⁶ moiety is attached to acarbon atom and is selected from H, halo, —OH, —C₁₋₆alkyl, and—O—C₁₋₆alkyl. In one embodiment, R⁶ is absent or is selected from H,halo (e.g., chloro and fluoro), —OH, and —C₁₋₆alkyl (e.g., —CH₃).

Specific examples of compounds of formula I include compounds offormulas Ic to Il, as described below. In one embodiment R² is —OR²⁰,R²⁰ is H, and R³ is Cl, which can be depicted as formula Ic:

In an exemplary embodiment of compounds of formula Ic, R¹ is —OR¹⁰; R¹⁰is H or —C₁₋₆alkyl; X is selected from furan, triazole, oxazole,isoxazole, pyridine, pyrazine, pyrimidine, benzotriazole,pyridylimidazole, pyridyltriazole, imidazopyridine,5-oxa-3,3a-diazacyclopenta[a]naphthalene, tetrahydropyridazine, anddihydrooxadiazabenzo[e]azulene; R⁴ is selected from H; halo;—C₀₋₅alkylene-OH; —C₁₋₆alkyl; —C₀₋₁alkylene-C(O)OR⁴¹; ═O; phenyloptionally substituted with one halo, and phenyl substituted with—C(O)OR⁴¹ when X is pyrazine or pyridine; R⁴¹ is H or —C₁₋₆alkyl; R⁵ isabsent or is selected from H, —C₀₋₃alkylene-OH,

R⁵³ is H or halo; R⁵⁴ is H or halo; R⁵⁵ is selected from H, halo, and—O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selected fromH, halo, —OH, and —C₁₋₆alkyl.

In another embodiment R² is —OR²⁰, R²⁰ is H, and R³ is —CF₃, which canbe depicted as formula Id:

In an exemplary embodiment of compounds of formula Id, R¹ is —OR¹⁰; R¹⁰is H or —C₁₋₆alkyl; X is pyridyltriazole; R⁴ is H; R⁵ is H; and R⁶ is H.

In still another embodiment R² is —OR²⁰; R²⁰ is H; and R³ is H, whichcan be depicted as formula Ie:

In an exemplary embodiment of compounds of formula Ie, R¹ is —OR¹⁰; R¹⁰is H or —C₁₋₆alkyl; X is selected from thiophene, imidazole, pyridine,pyrazine, pyridazine, benzimidazole, benzotriazole, pyridyltriazole,pyrrolopyrimidine, and hexahydropyrroloquinoxaline; R⁴ is selected fromH; halo; —C₁₋₆alkyl; —C₀₋₁alkylene-C(O)OR⁴¹; ═O; and phenyl substitutedwith —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine; R⁴¹ is H or—C₁₋₆alkyl; R⁵ is absent or is selected from H; —C₀₋₃alkylene-OH;—C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridineoptionally substituted with halo;

-   -   and

R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is H or —O—C₁₋₆alkyl; R⁵⁴ is H; R⁵⁵ isH v —O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selectedfrom H and halo.

In one embodiment R² is —OR²⁰, R²⁰ is H, and R³ is —CH₃, which can bedepicted as formula If:

In another embodiment R² is —OR²⁰, R²⁰ is H, and R³ is F, which can bedepicted as formula Ig:

In one embodiment R² is H and R³ is Cl, which can be depicted as formulaIh:

In an exemplary embodiment of compounds of formula Ih, R¹ is —OR¹⁰; R¹⁰is H or —C₁₋₆alkyl; X is selected from triazole, pyridine, pyrimidine,pyridazine, benzotriazole, and pyridyltriazole; R⁴ is selected from H,halo, —C₀₋₅alkylene-OH, —C₁₋₆alkyl, and phenyl substituted with—C(O)OR⁴¹ when X is pyridazine or pyridine; R⁴¹ is H; R⁵ is absent or isH; and R⁶ is absent or is H.

In another embodiment R² is H and R³ is —CF₃, which can be depicted asformula Ii:

In still another embodiment R² is H and R³ is H, which can be depictedas formula Ij:

In one embodiment R² is H and R³ is —CH₃, which can be depicted asformula Ik:

In another embodiment R² is H and R³ is F, which can be depicted asformula Il:

In yet another embodiment, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X isselected from thiophene, imidazole, triazole, oxazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, benzimidazole,benzotriazole, pyridylimidazole, pyridyltriazole, imidazopyridine,pyrrolopyrimidine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene,dihydrotriazole, dihydroisoxazole, tetrahydropyridazine,hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene; R⁴ isselected from H; halo; —C₀₋₅alkylene-OH; —C₁₋₆alkyl;—C₀₋₁alkylene-C(O)OR⁴¹; ═O; phenyl optionally substituted with one halo;and phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, orpyridine; R⁴¹ is selected from H and —C₁₋₆alkyl; R⁵ is absent or isselected from H; —C₀₋₃alkylene-OH; —C₁₋₃alkylene-C(O)OR⁵⁰;—CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted withhalo;

R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is selected from H, halo, and—O—C₁₋₆alkyl; R⁵⁴ is H or halo; R⁵⁵ is selected from H, halo, and—O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selected fromH, halo, —OH, and —C₁₋₆alkyl. In one embodiment, these compounds haveformulas Ic to Il. In still another compound, these compounds have R²being selected from H and —OR²⁰, where R²⁰ is H; and R³ being selectedfrom H, Cl, and —CF₃.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well aspharmaceutically acceptable salts thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.More specifically, the following abbreviations and reagents are used inthe schemes presented below:

P¹ represents a “carboxy-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at acarboxy group. Representative carboxy-protecting groups include, but arenot limited to, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), diphenylmethyl (benzhydryl, DPM) and thelike. Standard deprotection techniques and reagents are used to removethe P¹ group, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when P¹ is methyl,an acid such as TFA or HCl is commonly used when P¹ is ethyl or t-butyl,and H₂/Pd/C may be used when P¹ is benzyl.

P² represents an “amino-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at an aminogroup. Representative amino-protecting groups include, but are notlimited to, t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), and the like. Standard deprotectiontechniques are used to remove the P² group. For example, a BOC group canbe removed using an acidic reagent such as TFA in DCM or HCl in1,4-dioxane, while a Cbz group can be removed by employing catalytichydrogenation conditions such as H₂ (1 atm) and 10% Pd/C in an alcoholicsolvent (“H₂/Pd/C”).

P³ represents a “hydroxyl-protecting group,” a term that is used hereinto mean a protecting group suitable for preventing undesired reactionsat a hydroxyl group. Representative hydroxyl-protecting groups include,but are not limited to C₁₋₆alkyls, silyl groups includingtriC₁₋₆alkylsilyl groups, such as trimethylsilyl (TMS), triethylsilyl(TES), and tert-butyldimethylsilyl (TBDMS); esters (acyl groups)including C₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, andaromatic acyl groups such as benzoyl; arylmethyl groups such as benzyl(Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like. Standard deprotection techniques andreagents are used to remove the P³ group, and may vary depending uponwhich group is used. For example, H₂/Pd/C is commonly used when P³ isbenzyl, while NaOH is commonly used when P³ is an acyl group.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), 4-methylmorpholine, sodiumhydroxide, potassium hydroxide, potassium t-butoxide, and metalhydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying (forexample, over MgSO₄, over Na₂SO₄, or in vacuo); filtering; crystallizing(for example, from EtOAc and hexanes); being concentrated (for example,in vacuo); and/or purification (e.g., silica gel chromatography, flashchromatography, preparative HPLC, reverse phase-HPLC, orcrystallization).

Compounds of formula I, as well as their salts, can be prepared as shownin Scheme I:

Compound 1 is coupled with compound 2. In instances where R¹ is a groupsuch as —OCH₃ or —OCH₂CH₃, the coupling step may be followed by adeprotection step to provide a compound of formula I where R¹ is a groupsuch as —OH. Thus, one method of preparing compounds of the inventioninvolves coupling compound 1 and compound 2, with an optionaldeprotection step to form a compound of formula I or a pharmaceuticallyacceptable salt thereof.

Compounds of formula I and their salts, where R⁴ is optionallysubstituted phenyl, can also be prepared as shown in Scheme II:

Again, as with Scheme I, this is a standard coupling reaction betweencompound 1 and compound 2a to yield the compound 3, where L a leavinggroup such as halo (e.g., bromo and chloro). Compound 3 is then reactedwith optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane in apalladium-catalyzed coupling reaction. To prepare a compound of formulaI where R⁴ is unsubstituted phenyl, then R is hydrogen. Otherwise, R isselected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl. R may alsobe —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine. Exemplarysubstituted dioxaborolanes include 2-t-butoxycarbonylphenylboronic acidpinacol ester.

The palladium-catalyzed coupling reaction is typically conducted in aninert diluent in the presence of a suitable base such as potassiumcarbonate or sodium carbonate, and may be followed by a deprotectionstep to provide a compound of formula I where R¹ is a group such as —OH.Thus, another method of preparing compounds of the invention involvescoupling compound 1 and compound 2a to form compound 3, couplingcompound 3 with optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane, and an optionaldeprotection step to form a compound of formula I or a pharmaceuticallyacceptable salt thereof.

Compounds of formula I and their salts, where R⁴ is optionallysubstituted phenyl, can also be prepared as shown in Scheme III:

As with Scheme I, the first step is a palladium-catalyzed couplingreaction between compound 2b and an optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4,where L a leaving group such as halo (e.g., bromo and chloro) and P¹ isa carboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl,t-butyldimethylsilyl, and diphenylmethyl. Compound 4 is then coupledwith compound 1, followed by an optional deprotection step to provide acompound of formula I where R¹ is a group such as —OH. Thus, anothermethod of preparing compounds of the invention involves couplingcompound 2b with optionally substituted4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4,coupling compound 4 and compound 1, and an optional deprotection step toform a compound of formula I or a pharmaceutically acceptable saltthereof.

Compounds 1, 2, 2a, and 2b are generally commercially available or canbe prepared using procedures that are known in the art as well as thosethat are set forth in the Examples herein.

Certain intermediates described herein are believed to be novel andaccordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula II, or a saltthereof:

where P is selected from —O—P¹, —NHP², and —NH(O—P³); P¹ is acarboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl,t-butyldimethylsilyl, and diphenylmethyl; P² is an amino-protectinggroup selected from t-butoxycarbonyl, trityl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, andt-butyldimethylsilyl; and P³ is a hydroxyl-protecting group selectedfrom C₁₋₆alkyl, silyl groups, esters, and arylmethyl groups; and R²-R⁶are as defined for formula I. Thus, another method of preparingcompounds of the invention involves deprotecting a compound of formulaII.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess neprilysin (NEP) inhibition activity,that is, the compounds are able to inhibit enzyme-catalytic activity. Inanother embodiment, the compounds do not exhibit significant inhibitoryactivity of the angiotensin-converting enzyme. One measure of theability of a compound to inhibit NEP activity is the inhibition constant(pK_(i)). The pK_(i) value is the negative logarithm to base 10 of thedissociation constant (K_(i)), which is typically reported in molarunits. Compounds of the invention of particular interest are thosehaving a pK_(i) at NEP greater than or equal to 6.0, particularly thosehaving a pK_(i) greater than or equal to 7.0, and even more particularlythose having a pK_(i) greater than or equal to 8.0. In one embodiment,compounds of interest have a pK_(i) in the range of 6.0-6.9; in anotherembodiment, compounds of interest have a pK_(i) in the range of 7.0-7.9;in yet another embodiment, compounds of interest have a pK_(i) in therange of 8.0-8.9; and in still another embodiment, compounds of interesthave a pK_(i) in the range of greater than or equal to 9.0. Such valuescan be determined by techniques that are well known in the art, as wellas in the assays described herein.

Another measure of the ability of a compound to inhibit NEP activity isthe apparent inhibition constant (IC₅₀), which is the molarconcentration of compound that results in half-maximal inhibition ofsubstrate conversion by the NEP enzyme. The pIC₅₀ value is the negativelogarithm to base 10 of the IC₅₀. Compounds of the invention that are ofparticular interest, include those that exhibit a pIC₅₀ for NEP greaterthan or equal to about 5.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP ≧about 6.0 or a pIC₅₀ for NEP ≧about 7.0. Inanother embodiment, compounds of interest have a pIC₅₀ for NEP withinthe range of about 7.0-11.0; and in another embodiment, within the rangeof about 8.0-11.0, such as within the range of about 8.0-10.0.

It is noted that in some cases, compounds of the invention may possessweak NEP inhibition activity. In such cases, those of skill in the artwill recognize that these compounds still have utility as researchtools.

Exemplary assays to determine properties of compounds of the invention,such as the NEP inhibiting activity, are described in the Examples andinclude by way of illustration and not limitation, assays that measureNEP inhibition (described in Assay 1). Useful secondary assays includeassays to measure ACE inhibition (also described in Assay 1) andaminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005)JPET 315:1306-1313). A pharmacodynamic assay to assess the in vivoinhibitory potencies for ACE and NEP in anesthetized rats is describedin Assay 2 (see also Seymour et al. (1985) Hypertension 7(SupplI):I-35-I-42 and Wigle et al. (1992) Can. J Physiol. Pharmacol.70:1525-1528), where ACE inhibition is measured as the percentinhibition of the angiotensin I pressor response and NEP inhibition ismeasured as increased urinary cyclic guanosine 3′,5′-monophosphate(cGMP) output.

There are many in vivo assays that can be used to ascertain furtherutilities of the compounds of the invention. The conscious spontaneouslyhypertensive rat (SHR) model is a renin dependent hypertension model,and is described in Assay 3. See also Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity, and is described in Assay 4. See alsoTrapani et al. (1989) J Cardiovasc. Pharmacol. 14:419-424, Intengan etal. (1999) Hypertension 34(4):907-913, and Badyal et al. (2003) supra).The DOCA-salt model is particularly useful for evaluating the ability ofa test compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensitive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is describedin Assay 5. See also Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al. (1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hotplate, tail-flick and formalin tests, can be used to measure theanalgesic properties of compounds of the invention, as well as thespinal nerve ligation (SNL) model of neuropathic pain. See, for example,Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15.

Compounds of the invention are expected to inhibit the NEP enzyme in anyof the assays listed above, or assays of a similar nature. Thus, theaforementioned assays are useful in determining the therapeutic utilityof compounds of the invention, for example, their utility asantihypertensive agents or antidiarrheal agents. Other properties andutilities of compounds of the invention can be demonstrated using otherin vitro and in vivo assays well-known to those skilled in the art.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs may not exhibit the expected activityin an assay, but are expected to exhibit the desired activity oncemetabolized.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to NEP inhibition.Thus it is expected that patients suffering from a disease or disorderthat is treated by inhibiting the NEP enzyme or by increasing the levelsof its peptide substrates, can be treated by administering atherapeutically effective amount of a compound of the invention. Forexample, by inhibiting NEP, the compounds are expected to potentiate thebiological effects of endogenous peptides that are metabolized by NEP,such as the natriuretic peptides, bombesin, bradykinins, calcitonin,endothelins, enkephalins, neurotensin, substance P and vasoactiveintestinal peptide. Thus, these compounds are expected to have otherphysiological actions, for example, on the renal, central nervous,reproductive and gastrointestinal systems.

In one embodiment of the invention, patients suffering from a disease ordisorder that is treated by inhibiting the NEP enzyme, are treated byadministering a compound of the invention that is in its active form,i.e., a compound of formula I where R¹ is selected from —OR¹⁰ and—NR⁶⁰R⁷⁰, where R¹⁰ is H, R⁶⁰ is H or —OH, and R⁷⁰ is H; and R², R³, X,R⁴, R⁵, and R⁶ are as defined for formula I.

In another embodiment, patients are treated by administering a compoundthat is metabolized in vitro to form a compound of formula I, where R¹is —OR¹⁰ or —NR⁶⁰R⁷⁰; R¹⁰ is H, R⁶⁰ is H or —OH, R⁷⁰ is H; and R², R³,X, R⁴, R⁵, and R⁶ are as defined for formula I.

In another embodiment, patients are treated by administering a compoundof the invention that is in its prodrug form at the R¹ group, i.e., acompound of formula I where:

R¹ is —OR¹⁰; and R¹⁰ is selected from —C₁₋₆alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵,—C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl,pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is H; or

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl,pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹; or

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is H or —OH; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹; or

R¹ is —OR¹⁰; R² is —OR²⁰; and R²⁰ is taken together with R¹⁰ to form—CR²¹R²²—; or

R¹ is —NR⁶⁰R⁷⁰; R² is —OR²⁰; and R²⁰ is taken together with R⁶⁰ to form—C(O)—; and R², R³, X, R⁴, R⁵, R⁶, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²¹, R²²,R⁶¹, R⁶², R⁶³, and R⁷¹ are as defined for formula I.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Roques et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure, with or without accompanying renal disease. Thus, oneembodiment of the invention relates to a method for treatinghypertension, particularly primary hypertension or pulmonary arterialhypertension, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, aldosterone synthase inhibitors,angiotensin-converting enzyme inhibitors and dual-actingangiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/al-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a calciumchannel blocker, a diuretic, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease. Whenused to treat resistant hypertension, the compound may be administeredin combination with other therapeutic agents such as aldosteronesynthase inhibitors.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergic receptorantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarçais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal agents.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional antiglaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Roques et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, the compounds of the invention maybe combined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005) J. Investigative Med. 53:360-365), depression (see Nobleet al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since compounds of the invention possess NEP enzyme inhibition activity,such compounds are also useful as research tools for investigating orstudying biological systems or samples having a NEP enzyme, for exampleto study diseases where the NEP enzyme or its peptide substrates plays arole. Any suitable biological system or sample having a NEP enzyme maybe employed in such studies which may be conducted either in vitro or invivo. Representative biological systems or samples suitable for suchstudies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, isolated organs, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), andthe like, with mammals being of particular interest. In one particularembodiment of the invention, NEP enzyme activity in a mammal isinhibited by administering a NEP-inhibiting amount of a compound of theinvention. Compounds of the invention can also be used as research toolsby conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering the compoundto a mammal, for example by i.p., p.o, i.v., s.c., or inhaledadministration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asenzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingNEP-inhibiting activity. In this manner, a compound of the invention isused as a standard in an assay to allow comparison of the resultsobtained with a test compound and with compounds of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to a compound of the invention, if any. This aspect ofthe invention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages that may be present inthe compound. These formulations may be rendered sterile by use of asterile injectable medium, a sterilizing agent, filtration, irradiation,or heat. In one particular embodiment, the parenteral formulationcomprises an aqueous cyclodextrin solution as the pharmaceuticallyacceptable carrier. Suitable cyclodextrins include cyclic moleculescontaining six or more α-D-glucopyranose units linked at the 1,4positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzyme/neprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like Typically, theβ₂-adrenoreceptor agonist will be administered in an amount sufficientto provide from about 0.05-500 μg per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltopril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof.

In a particular embodiment, the ACE inhibitor is selected from:benazepril, captopril, enalapril, lisinopril, ramipril, and combinationsthereof. Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day. In anotherembodiment, compounds of the invention are administered in combinationwith a dual-acting angiotensin-converting enzyme/neprilysin (ACE/NEP)inhibitor, examples of which include, but are not limited to: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth sabsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockerssuch as betaxolol, levobunolol, and timolol; carbonic anhydraseinhibitors such as acetazolamide, brinzolamide, or dorzolamide;cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrinecompounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to: aspirin;anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine;heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, irbesartan, saprisartan, tasosartan, telmisartan,valsartan, and combinations thereof. Exemplary salts and/or prodrugsinclude candesartan cilexetil, eprosartan mesylate, losartan potassiumsalt, and olmesartan medoxomil. Typically, the ARB will be administeredin an amount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonist/neprilysininhibitor (ARB/NEP) inhibitor, examples of which include, but are notlimited to, compounds described in U.S. Publication Nos. 2008/0269305and 2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, suchas the compound,4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, bamidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide(NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof.

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155(N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(5)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid), which have activity both as inhibitors of the endothelinconverting enzyme (ECE) and of NEP. Other dual acting ECE/NEP compoundscan also be used. The NEP inhibitor will be administered in an amountsufficient to provide from about 20-800 mg per day, with typical dailydosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,amoxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lomoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptanrizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrignine, lidocaine, mexiletine, oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-3-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated. The following abbreviations have thefollowing meanings unless otherwise indicated and any otherabbreviations used herein and not defined have their standard, generallyaccepted meaning:

-   -   AcOH acetic acid    -   Cbz carbobenzyloxy (—C(O)O-benzyl)    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMF N,N-dimethylformamide    -   Dnp 2,4-dinitrophenyl    -   EDCI N-(3-dimethylaminopropyl)-N″-ethylcarbodiimide    -   EtOAc ethyl acetate    -   EtOH ethanol    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   HOBt 1-hydroxybenzotriazole hydrate    -   Mca (7-methoxycoumarin-4-yl)acyl    -   MeCN acetonitrile    -   MeOH methanol    -   Pd(PPh₃)₄ tetrakis(triphenylphosphine)palladium(0)    -   SilicaCat® DPP-Pd silica based diphenylphosphine palladium (II)        catalyst    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haen, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationfor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1 [(R)-1-(2-Chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamicAcid Benzyl Ester

To a suspension of (R)-2-amino-3-(2-chloro-phenyl)-propionic acid (100.0g, 0.5 mol) in water (1 L) was added dropwise 4N aqueous NaOH (125 mL)at 0° C. Then a solution of N-(benzyloxycarbonyloxy)succinimide (125.0g, 0.5 mol) in acetone (300 mL) was added in one portion. The pH of themixture was maintained at 8-9 by addition of 3N aqueous NaOH. After themixture was stirred for 4 hours, the pH was adjusted to 1 with 6N HCland the mixture was extracted with EtOAc (2×500 mL). The combinedextracts were washed with 1N HCl (2×500 mL), dried over anhydrous Na₂SO₄and concentrated to yield compound (1) as a white solid (155.0 g).

To a solution of compound (1) (80.0 g, 240 μmol) in DCM (500 mL) wasadded EDCI (50.6 g, 264 μmol), HOBt (35.6 g, 264 μmol),N,O-dimethylhydroxylamine hydrochloride (51.5 g, 528 μmol) andtriethylamine (111 mL, 790 μmol). The mixture was stirred at roomtemperature overnight. Then the mixture was washed with 2N HCl (3×500mL) and saturated aqueous NaHCO₃ (3×500 mL), respectively. The organiclayer was dried over anhydrous Na₂SO₄ and concentrated to yield compound(2) as a yellow oil (71.0 g).

To a suspension of LiAl H₄ (7.2 g, 188 μmol) in THF (800 mL) was addeddropwise a solution of compound (2) (71.0 g, 188 μmol) in THF (200 mL)at −20° C. The mixture was stirred at −20° C. for 2 hours. Then thereaction was quenched carefully with 1N HCl. The mixture was extractedwith EtOAc (2×600 mL) and the combined extracts were dried overanhydrous Na₂SO₄ and concentrated to yield compound (3) (59.0 g), whichwas used directly without further purification.

To a solution of compound (3) (59.0 g, 188 μmol) in THF (500 mL) wasadded aqueous NaHSO₃ (19.5 g in 500 mL of water) and the mixture wasstirred at 0° C. overnight. NaCN (9.2 g, 188 μmol) was added and theresulting mixture was stirred for 3 hours. The mixture was extractedwith EtOAc (2×500 mL) and the combined extracts were dried overanhydrous Na₂SO₄ and concentrated to yield the title compound (64.0 g),which was used directly without further purification.

Preparation 2 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric acidMethyl Ester

A mixture of [(R)-1-(2-chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamicacid benzyl ester (55.0 g, 157 μmol) in dioxane (300 mL) and 6N HCl (300mL) was heated under reflux overnight. The solvent was removed underreduced pressure and the residue was dissolved in a 3N HCl-MeOHsolution. The resulting mixture was refluxed for 4 hours and the solventwas removed under reduced pressure. The residue was taken up in EtOAc(500 mL) and aqueous NaHCO₃ (500 mL). The organic layer was separated,dried over anhydrous Na₂SO₄ and concentrated to give a mixture of thetitle compound and its (R,S)-isomer, which was subjected to flash columnchromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (8.1g).

¹H NMR (CDCl₃): δ 7.37 (m, 1H), 7.22 (m, 3H), 4.30 (d, J=3.3 Hz, 1H),3.80 (s, 3H), 3.51 (m, 1H), 2.96 (m, 1H), 2.71 (m, 1H), 2.06 (br s, 2H).MS (m/z): 244 [M+H]⁺.

Preparation 3 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric AcidEthyl Ester

A mixture of [(R)-1-(2-chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamicacid benzyl ester (64.0 g, 188 μmol) in dioxane (300 mL) and 6N HCl (300mL) was heated under reflux overnight. The solvent was removed underreduced pressure and the residue was dissolved in a 3N HCl-EtOHsolution. The resulting mixture was refluxed for 4 hours and the solventwas removed under reduced pressure. The residue was taken up in EtOAc(500 mL) and aqueous NaHCO₃ (500 mL). The organic layer was separated,dried over anhydrous Na₂SO₄ and concentrated to give a mixture of thetitle compound and its (R,S)-isomer, which was subjected to flash columnchromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (9.7g).

¹H NMR (CDCl₃): δ 7.37 (m, 1H), 7.22 (m, 3H), 4.25 (m, 3H), 3.50 (s,1H), 2.97 (d, J=10.8 Hz, 1H), 2.74 (t, J=11.4 Hz, 1H), 2.06 (br s, 2H),1.35 (t, J=7.1 Hz, 3H). MS (m/z): 258 [M+H]⁺.

Example 1

2-{2-(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl-pyridin-4-yl}-benzoicacid (R¹=—OH: R⁴¹=H)

Methyl 4-bromopicolinate (167 mg, 772 μmol. 1.0 eq.) was mixed with2-t-butoxycarbonylphenylboronic acid pinacol ester (282 mg, 927 μmol,1.2 eq.) in toluene (1.1 mL). MeOH (409 μL) was added, followed by K₂CO₃(214 mg, 1.5 mmol, 2.0 eq.) predissolved in water (202 μL). The mixturewas stirred, and the reaction vessel was capped, placed under vacuum andpurged with nitrogen. Pd(PPh₃)₄(89.2 mg, 77.2 μmol) was added. Thevessel was recapped and heated at 110° C. for 50 minutes. The organiclayer was removed and the product was evaporated under vacuum. THF (3mL) and 1 M aqueous NaOH (1.5 mL, 2.0 eq.) was added and the mixture wasstirred for 2 hours. EtOAc was added and mixture was acidified withsaturated NH₄Cl. The organic layers were extracted, dried and evaporatedto yield 4-(2-t-butoxycarbonyl-phenyl)-pyridine-2-carboxylic acid.

4-(2-t-Butoxycarbonyl-phenyl)-pyridine-2-carboxylic acid (30 mg, 0.1mmol, 1.0 eq.) and (2R,3R)-3-amino-4-(2-chloro-phenyl)-2-hydroxy-butyricacid ethyl ester (16.8 mg, 65.1 μmol, 1.0 eq.) were combined with DIPEA(52.4 μL, 3.0 eq.) and HATU (26.7 mg, 70.2 μmol, 0.7 eq.) and DCM (0.8mL), and the resulting mixture was stirred for 45 minutes at roomtemperature. The reaction was quenched with saturated NH₄Cl, and theproduct was extracted with DCM, dried and evaporated to yield2-{2-[(R,2R)-1-(2-chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoicacid t-butyl ester.

2-{2-[(R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoicacid t-butyl ester (104 mg, 192 μmol, 1.0 eq.) was combined with 1:1TFA/DCM (1 mL each) and the resulting mixture was stirred for 1 hour.The solvent was evaporated and THF (3 mL) and 1 M aqueous NaOH (577 μL,3.0 eq.) were added and the resulting mixture was stirred for 2 hours.The solvent was evaporated and AcOH (2 mL) was added. The product wasthen purified by preparative HPLC to yield the title compound as a TFAsalt (2.4 mg, 96% purity). MS m/z [M+H]⁺ calc'd for C₂₃H₁₉ClN₂O₆,455.09. found 455.

2-{2-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoicacid ethyl ester (R¹=—OCH₂CH₃; R⁴¹=—CH₂CH₃)

4-Bromopyridine-2-carboxylic acid (93.9 mg, 465 μmol, 1.0 eq.) and(2R,3R)-3-amino-4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl ester(120 mg, 465 μmol, 1.0 eq.) were combined with DIPEA (434 μL) and HATU(177 mg, 465 μmol, 1.0 eq.) in DCM (4 mL) and stirred for 45 minutes atroom temperature. The reaction was quenched with saturated NaHCO₃ andextracted with DCM, dried and evaporated to yield(2R,3R)-3-[(4-bromo-pyridine-2-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyricacid ethyl ester. This crude product was mixed with ethyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (154 mg, 558μmol, 1.2 eq.) and toluene (540 μL). EtOH (0.3 mL) was added, followedby K₂CO₃ (128 mg, 930 μmol, 2.0 eq.) predissolved in water (0.1 mL). Themixture was stirred, and the reaction vessel was capped, placed undervacuum and purged with nitrogen. Pd(PPh₃)₄(53.7 mg, 46.5 μmol) wasadded. The vessel was recapped and heated at 100° C. for 30 minutes. Theorganic layer was retained and the product was evaporated under vacuumthen purified using preparative HPLC to yield the title compound as aTFA salt (30 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₇H₂₇ClN₂O₆,511.16. found 512.2.

2-{2-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoicacid (R¹=—OCH₂CH₃; R⁴¹=H)

Crude2-{2-[(1R,2R)-1-(2-chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoicacid ethyl ester was dissolved in DCM/TFA (0.3 mL each) and stirred for2 hours. The solvent was evaporated and the product was purified usingpreparative HPLC to yield the title compound as a TFA salt (75 mg, 97%purity). MS m/z [M+H]⁺ calc'd for C₂₅H₂₃ClN₂O₆, 483.12. found 483.

Example 2

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula Ic, were prepared as the parent compound or as a TFAsalt:

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 1 OH

CH₃ C₂₄H₂₁ClN₂O₆ 469.11 469.0

-   1.    2-{2-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-6-methyl-pyridin-4-yl}-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 2 OH

H C₂₃H₁₉ClN₂O₆ 455.09 456 3 OCH₂CH₃

H C₂₇H₂₇ClN₂O₆ 511.16 511 4 OH

CH₃ C₂₄H₂₁ClN₂O₆ 469.11 470

-   2.    2-{4-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic    acid (TFA salt)-   3.    2-{4-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic    acid ethyl ester (TFA salt)-   4.    2-{4-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-6-methyl-pyridin-2-yl}-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 5 OH

H C₂₃H₁₉ClN₂O₆ 455.09 455.2 6 OCH₂CH₃

H C₂₅H₂₃ClN₂O₆ 483.12 483.2 7 OH

OH C₂₃H₁₉ClN₂O₇ 471.09 471.0

-   5.    2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-3-yl}-benzoic    acid (TFA salt)-   6. 2-{5-[(R,    2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-3-yl}-benzoic    acid (TFA salt)-   7.    2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-2-hydroxy-pyridin-3-yl}-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found  8 OH

H C₂₃H₁₉ClN₂O₆ 455.09 455    9 OH

Cl C₂₃H₁₈Cl₂N₂O₆ 489.05 490   10 OH

F C₂₃H₁₈ClFN₂O₆ 473.08 473.0

-   8.    2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic    acid (TFA salt)-   9.    2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3-chloro-pyridin-2-yl}-benzoic    acid (TFA salt)-   10.    2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3-fluoro-pyridin-2-yl}-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 11 OH OH C₁₄H₁₃ClN₂O₆341.05 341.0 12 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 436.0 13 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.4 14 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.2

-   11.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-butyric    acid (TFA salt)-   12.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(2-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)-   13.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(3-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)-   14.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(4-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 15 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.2

-   15.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[5-(2-chloro-phenyl)-isoxazole-3-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 16 OH ═O

C₂₁H₁₇Cl₂FN₂O₆ 483.04 483.2 17 OH ═O

C₂₂H₁₉ClF₂N₂O₇ 497.08 497.4

-   16.    (2R,3R)-3-{[2-(4-Chloro-2-fluoro-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)-   17.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[2-(2,3-difluoro-4-methoxy-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 18 OH

C₂₀H₁₇ClN₂O₅ 401.08 401.2

-   18.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(2-phenyl-oxazole-5-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 19 OH H C₁₃H₁₃ClN₄O₄325.06 325.0

-   19.    (2S,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 20 OH

C₂₀H₁₉ClN₄O₄ 415.11 415.2 21 OH OH C₁₃H₁₃ClN₄O₅ 341.06 341.0

-   20.    (2R,3R)-3-[(1-Benzyl-1H-[1,2,3]triazole-4-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)-   21.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 22 OH CH₃

C₂₀H₁₈Cl₂N₄O₄ 449.07 450.0

-   22.    (2R,3R)-4-(2-Chloro-phenyl)-3-{[1-(4-chloro-phenyl)-5-methyl-1H-[1,2,4]triazole-3-carbonyl]-amino}-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 23 OH ═O

C₂₀H₁₇Cl₂FN₄O₅ 483.06 483.4

-   23.    (2R,3R)-3-{[1-(4-Chloro-2-fluoro-benzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl]-amino}-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 24 OH C₂₁H₁₈ClN₃O₅ 428.09428.3 25 OCH₂CH₃ C₂₃H₂₂ClN₃O₅ 456.12 456.2

-   24.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(5-oxa-3,3a-diazacyclopenta[a]naphthalene-2-carbonyl)-amino]-butyric    acid (TFA salt)-   25.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(5-oxa-3,3a-diazacyclopenta[a]naphthalene-2-carbonyl)-amino]-butyric    acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 26 OH C₂₂H₂₀ClN₃O₅ 442.11442.4

-   26.    (2R,3R)-4-(2-Chloro-phenyl)-3-[(4,5-dihydro-6-oxa-3,3a-diaza-benzo[e]azulene-2-carbonyl)-amino]-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 27 OH F C₁₇H₁₄ClFN₄O₄393.07 393.0 28 OH Cl C₁₇H₁₄Cl₂N₄O₄ 409.04 410.0 29 OH H C₁₇H₁₅ClN₄O₄375.08 375.0 30 OH CH₃ C₁₈H₁₇ClN₄O₄ 389.09 389.0

-   27.    (2S,3R)-4-(2-Chloro-phenyl)-3-[(7-fluoro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-butyric    acid (TFA salt)-   28.    (2S,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)-   29.    (2S,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)-   30.    (2S,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 31 OH COOH C₁₈H₁₅ClN₄O₆419.07 419.2

-   31.    6-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3H-benzotriazole-4-carboxylic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 32 OH COOH C₁₆H₁₄ClNO₇368.05 368.0

-   32.    5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-furan-2-carboxylic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 33 OH Br C₁₈H₁₅BrClN₃O₄451.99 452.0

-   33.    (2R,3R)-3-[(3-Bromo-imidazo[1,2-a]pyridine-6-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 34 OH OH C₁₅H₁₄ClN₃O₅352.06 352.2

-   34.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(5-hydroxy-pyrazine-2-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 35 OH OH C₁₅H₁₄ClN₃O₅352.06 352.4

-   35.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 36 OH C₁₇H₁₅ClN₄O₄ 375.08375.0

-   36.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3H-imidazo[4,5-b]pyridine-6-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 37 OH C₁₆H₁₄ClN₅O₄ 376.07n.d. 38 OCH₂CH₃ C₁₈H₈ClN₅O₄ 404.10 404.2 n.d. = not determined

-   37.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric    acid (TFA salt)-   38.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric    acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 39 OH ═O C₁₅H₁₆ClN₃O₅354.08 354.0

-   39.    (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(6-oxo-1,4,5,6-tetrahydro-pyridazine-3-carbonyl)-amino]-butyric    acid

Preparation 4(2R,3R)-3-Amino-2-hydroxy-4-(2-trifluoromethyl-phenyl)-butyric AcidEthyl Ester

To a suspension of (R)-2-amino-3-(2-trifluoromethyl-phenyl)-propionicacid (25.0 g, 107 μmol) in water (230 mL) was added dropwise 4N aqueousNaOH (25 mL) at 0° C. Then a solution ofN-(benzyloxycarbonyloxy)succinimide (26.8 g, 107 μmol) in acetone (100mL) was added in one portion. The pH of the mixture was maintained at8-9 by addition of 3N aqueous NaOH. After the mixture was stirred for 4hours, the pH was adjusted to 1 with 6N HCl and the mixture wasextracted with EtOAc (2×200 mL). The combined extracts were washed with1N HCl (2×200 mL), dried over anhydrous Na₂SO₄ and concentrated to yieldcompound (1) as a white solid (35.0 g).

To a solution of compound (1) (35.0 g, 95 μmol) in DCM (300 mL) wasadded EDCI (20.0 g, 104 μmol), HOBt (14.0 g, 104 μmol),N,O-dimethylhydroxylamine hydrochloride (20.0 g, 208 μmol) andtriethylamine (33.0 g, 312 μmol). The mixture was stirred at roomtemperature overnight. Then the mixture was washed with 2N HCl (3×250mL) and saturated aqueous NaHCO₃ (3×200 mL), respectively. The organiclayer was dried over anhydrous Na₂SO₄ and concentrated to yield compound(2) (35.0 g).

To a suspension of LiAl H₄ (3.3 g, 85 μmol) in THF (300 mL) was addeddropwise a solution of compound (2) (35.0 g, 85 μmol) in THF (150 mL) at−20° C. The mixture was stirred at −20° C. for 2.5 hours. Then thereaction was quenched carefully with 1N HCl. The mixture was extractedwith EtOAc (2×300 mL) and the combined extracts were dried overanhydrous Na₂SO₄ and concentrated to yield compound (3) (29.8 g), whichwas used directly without further purification.

To a solution of compound (3) (29.8 g, 85 μmol) in THF (250 mL) wasadded aqueous NaHSO₃ (8.84 g in 250 mL of water) and the mixture wasstirred at 0° C. overnight. NaCN (4.2 g, 85 μmol) was added and theresulting mixture was stirred for 3 hours. The mixture was extractedwith EtOAc (2×300 mL) and the combined extracts were dried overanhydrous Na₂SO₄ and concentrated to yield compound (4) (32.1 g,quantitative), which was used directly without further purification.

A mixture of compound (4) (32.1 g, 85 μmol) in dioxane (200 mL) and 6NHCl (200 mL) was heated under reflux overnight. The solvent was removedunder reduced pressure and the residue was dissolved in a 3N HCl-EtOHsolution. The resulting mixture was refluxed overnight and the solventwas removed under reduced pressure. The residue was taken up in EtOAc(300 mL) and aqueous NaHCO₃ (300 mL). The organic layer was separated,dried over anhydrous Na₂SO₄ and concentrated to give a mixture of thetitle compound and its (R,S)-isomer, which was subjected to flash columnchromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (6.0g).

¹H NMR (CDCl₃): δ 7.68 (d, J=7.8 Hz, 1H), 7.52 (t, J=7.5 Hz, 1H), 7.37(m, 2H), 4.31 (m, 3H), 3.42 (m, 1H), 3.00 (dd, J=2.4, 14.4 Hz, 1H), 2.79(m, 1H), 1.37 (t, J=7.1 Hz, 3H). MS (m/z): 292 [M+H]⁺.

Preparation 5 1H-[1,2,3]Triazolo[4,5-b]pyridine-6-carboxylic Acid

Methyl 6-amino-5-nitronicotinate (5.0 g, 25.4 mmol, 1.0 eq.) wascombined with acetic anhydride (100 g) and sulfuric acid (1 mL). Theresulting solution was stirred for 2 hours at room temperature. Thesolids were collected by filtration and washed with 10% NaHCO₃ (aq.).The solid was dried in an oven under reduced pressure to yield compound(1) as a yellow solid (3.9 g).

Compound (1) (3.9 g, 16.3 mmol, 1.0 eq.) was dissolved in MeOH (200 mL),and palladium carbon (0.5 g) was added. The resulting solution wasstirred overnight at room temperature, under hydrogen. The solids werefiltered out and the resulting mixture was concentrated under vacuum toyield compound (2) as a white solid (3.0 g).

Compound (2) (3.0 g, 14.4 mmol, 1.0 equiv) was dissolved in sulfuricacid/H₂O (1:4) (100 mL). A solution of NaNO₂ (1.1 g, 15.7 mmol, 1.1 eq.)in water (15 mL) was added dropwise with stirring at −5˜0 CC over 5minutes. The resulting solution was stirred for 1 hour at 0° C. in awater/ice bath. The solids were collected by filtration and washed withwater. The solid was dried in an oven under reduced pressure to yieldcompound (3) as a yellow solid (2.5 g).

Compound (3) (3.5 g, 19.7 mmol, 1.0 eq.) was dissolved in THF (20 mL). Asolution of NaOH (5.6 g, 140.0 mmol, 7.1 eq. in water (50 mL) was added,and the resulting solution was stirred for 4 hours at room temperature.The resulting mixture was concentrated under vacuum, then washed withEtOAc (2×30 mL). The pH value of the solution was adjusted to 3 with HCl(5 mol/L). The solids were collected by filtration, and dried in an ovenunder reduced pressure to yield the title compound as a white solid (2.7g). ES, m/z: 165 [M+H]+; ¹HNMR (DMSO, 300 Hz, ppm) 13.70 (s, 1H), 9.19(s, 1H), 8.92 (s, 1H).

Example 3(2R,3R)-2-Hydroxy-3-[(3H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-4-(2-trifluoromethyl-phenyl)-butyricacid

1H-[1,2,3]Triazolo[4,5-b]pyridine-6-carboxylic acid (86 mg, 520 μmmol,1.0 eq.) was combined with HATU (239 mg, 629 μmol, 1.2 eq.) in DMF (3mL) and stirred at room temperature for 15 minutes.(2R,3R)-3-Amino-2-hydroxy-4-(2-trifluoromethyl-phenyl)-butyric acidethyl ester (153 mg, 524 μmol, 1.0 eq.) in DIPEA (182 μL, 2.0 eq.) andDMF (3 mL) was added and the resulting mixture was stirred overnight atroom temperature. 2 M aqueous NaOH (3 mL) and THF (3 mL) were added andthe mixture was heated at 40° C. overnight. The product was purified bypreparative HPLC to yield the title compound as a TFA salt (68.8 mg,100% purity). MS m/z [M+H]⁺ calc'd for C₁₇H₁₄F₃N₅O₄, 410.10. found410.2.

Example 4

2-[6-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicAcid (R¹=—OH; R⁴¹=H)

6-Chloropyridazine-3-carboxylic acid (78.9 mg, 497 μmol, 1.0 eq.), DIPEA(273 μL, 3.2 eq.), HATU (189 mg, 497 μmol, 1.0 eq.), and(2R,3R)-3-amino-2-hydroxy-4-phenyl-butyric acid ethyl ester (129 mg, 497μmol, 1.0 eq.) were combined in DCM (2 mL) and stirred for 1 hour. Thecrude reaction was chromatographed using a gradient (0-80% EtOAc/Hex) toobtain(2R,3R)-3-[(6-chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyricacid ethyl ester.

(2R,3R)-3-[(6-Chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyricacid ethyl ester (172 mg, 474 μmol. 1.0 eq.) was mixed with2-t-butoxycarbonylphenylboronic acid pinacol ester (173 mg, 568 μmol,1.2 eq.) in toluene (661 μL). EtOH (361 μL) was added, followed by K₂CO₃(131 mg, 947 μmol, 2.0 eq.) predissolved in water (124 μL). The mixturewas stirred, and the reaction vessel was capped, placed under vacuum andpurged with nitrogen. Pd(PPh₃)₄(54.7 mg, 47.4 μmol) was added. Thevessel was recapped and heated at 100° C. for 30 minutes. The organiclayer was removed and the product was evaporated under vacuum to yield2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicacid t-butyl ester.

2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicacid t-butyl ester (30 mg, 59.3 μmol) was combined with TFA and DCM 1:1(0.5 mL each) and the resulting mixture was stirred for 30 minutes. Thesolvent was evaporated and the product was dissolved in THF (0.5 mL). 5M NaOH (0.3 mL) was added and the mixture was stirred overnight. AcOHwas added to achieve a pH of 5 (˜1 mL). The product was then purified bypreparative HPLC to yield the title compound (1 mg, 98% purity). MS m/z[M+H]⁺ calc'd for C₂₂H₁₉N₃O₆, 422.13. found 422.

2-[6-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicAcid (R¹=—OCH₂CH₃; R⁴¹=H)

2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicacid t-butyl ester (97.3 mg, 192 μmol) was combined with TFA and DCM 1:1(1 mL each) and the resulting mixture was stirred for 1 hour. Thesolvent was evaporated and THF (3 mL) and 1 M aqueous NaOH (577 μL, 3.0eq.) were added. The mixture was stirred for 2 hours and EtOAc wasadded. The aqueous layer was extracted out and the organic was washedwith NaOH (0.2 mL), stirred and the aqueous layer extracted and added tothe first fraction. The aqueous was acidified to pH ˜5 with concentratedHCl, forming a gummy solid. The aqueous was removed and the solid wasdissolved in AcOH and purified by preparative HPLC to yield the titlecompound (15 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₄H₂₃N₃O₆,450.16. found 450.2.

2-[6-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicAcid Ethyl Ester (R¹=—OCH₂CH₃; R⁴¹=—CH₇CH₃)

6-Chloropyridazine-3-carboxylic acid (71 mg, 440 μmol, 1.0 eq.), K₂CO₃(185 mg, 1.3 μmol, 3.0 eq.), and2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzoic acid ethylester (148 mg, 535 μmol, 1.2 eq.) were combined with EtOH (1 mL) andwater (0.3 mL). The mixture was stirred, and the reaction vessel wascapped, placed under vacuum and purged with nitrogen. SilicaCat® DPP-Pd(280 μmol/g loading; 286 mg, 80.2 μmol) was added. The vessel wasrecapped and microwaved at 100° C. for 20 minutes. The solvent wasremoved and the product filtered. The pH was adjusted to ˜4 with 1N HCl.HATU (136 mg, 356 μmol, 0.8 eq.), DIPEA (233 μL, 3.0 eq.), and(2R,3R)-3-amino-2-hydroxy-4-phenyl-butyric acid ethyl ester (99.5 mg,446 μmol, 1.0 eq.) were combined in DCM (2 mL) and stirred for 2 hours.AcOH was added and the product was purified by preparative HPLC to yieldthe title compound as a TFA salt (1.8 mg, 95% purity). MS m/z [M+H]⁺calc'd for C₂₆H₂₇N₃O₆, 478.19. found 478.

2-[6-((1R,2R)-1-Benzyl-2-butoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoicAcid (R¹=—O(CH₂)₃CH₃; R⁴¹=H)

(2R,3R)-3-t-Butoxycarbonylamino-2-hydroxy-4-phenyl-butyric acid (70.9mg, 240 μmol, 1.0 eq.) and 1-butanol (2 mL, 20 mmol) were combined in 4M HCl in 1,4-dioxane (0.4 mL, 2 mmol), and stirred for 2 hours at 60° C.The solvent was evaporated and the reaction mixture was azeotroped withtoluene. 6-Chloropyridazine-3-carboxylic acid (38.1 mg, 240 μmol, 1.0eq.), DIPEA (134 μL, 3.2 eq.), and HATU (91.3 mg, 240 μmol, 1.0 eq.)were combined in DCM (5 mL) and stirred for 5 minutes at roomtemperature. This mixture was then added to the azeotroped mixture andstirred for 1 hour. The reaction was quenched with saturated NH₄Cl. Theproduct was extracted with DCM, dried and evaporated, then purified bychromatography (0-60% EtOAc/hexanes gradient) to yield(2R,3R)-3-[(6-chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyricacid butyl ester.

(2R,3R)-3-[(6-Chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyricacid butyl ester (90 mg, 230 μmol, 1.0 eq.) was mixed with2-t-butoxycarbonylphenylboronic acid pinacol ester (83.8 mg, 276 μmol,1.2 eq.) in toluene (320 μL). 1-Butanol (274 μL) was added, followed byK₂CO₃ (63.5 mg, 459 μmol) predissolved in water (60 μL). The mixture wasstirred, and the reaction vessel was capped, placed under vacuum andpurged with nitrogen. Pd(PPh₃)₄(26.5 mg, 23 μmol) was added. The vesselwas recapped and microwaved at 100° C. for 40 minutes. The organic layerwas removed, AcOH was added, and the product was purified by preparativeHPLC to yield the title compound as a TFA salt (41 mg, 95% purity). MSm/z [M+H]⁺ calc'd for C₂₆H₂₇N₃O₆, 478.19. found 478.2.

Example 5

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula Ie, were prepared as the parent compound or as a TFAsalt:

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 1 OH ═O H C₂₂H₂₃N₃O₅410.16 410.2

-   1.    (2R,3R)-2-Hydroxy-3-[(4-oxo-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-a]quinoxaline-8-carbonyl)-amino]-4-phenyl-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 2 OH (CH₂)₂—CH₃

CH₃ C₂₈H₂₉BrN₄O₄ 565.14 565.6 3 OH (CH₂)₂—CH₃

CH₃ C₂₈H₃₀N₄O₄ 487.23 487.6 4 OCH₂—CH₃ (CH₂)₂—CH₃

CH₃ C₃₀H₃₄N₄O₄ 515.26 515.4 5 OH (CH₂)₂—CH₃ CH₂COOH CH₃ C₂₄H₂₇N₃O₆454.19 454.2 6 OH (CH₂)₂—CH₃

CH₃ C₃₀H₃₃N₃O₅ 516.24 516.2 7 OH (CH₂)₂—CH₃

CH₃ C₃₀H₃₃N₃O₅ 516.24 516.2 8 OH (CH₂)₂—CH₃ CH₂—CONH₂ CH₃ C₂₄H₂₈N₄O₅453.21 453.2 9 OH CH₂—CH₃ H Cl C₂₀H₂₀ClN₃O₄ 402.11 402.0

-   2.    (2R,3R)-3-{[3-(6-Bromo-pyridin-3-ylmethyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-2-hydroxy-4-phenyl-butyric    acid (TFA salt)-   3.    (2R,3R)-2-Hydroxy-3-[(7-methyl-2-propyl-3-pyridin-3-ylmethyl-3H-benzoimidazole-5-carbonyl)-amino]-4-phenyl-butyric    acid (TFA salt)-   4.    (2R,3R)-2-Hydroxy-3-[(7-methyl-2-propyl-3-pyridin-3-ylmethyl-3H-benzoimidazole-5-carbonyl)-amino]-4-phenyl-butyric    acid ethyl ester (TFA salt)-   5.    (2R,3R)-3-[(3-Carboxymethyl-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid (TFA salt)-   6.    (2R,3R)-2-Hydroxy-3-{[3-(2-methoxy-benzyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-4-phenyl-butyric    acid (TFA salt)-   7.    (2R,3R)-2-Hydroxy-3-{[3-(4-methoxy-benzyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-4-phenyl-butyric    acid (TFA salt)-   8.    (2R,3R)-3-[(3-Carbamoylmethyl-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid (TFA salt)-   9.    (2R,3R)-3-[(7-Chloro-2-ethyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 10 OCH₂CH₃ CH₂CH₃ HCl C₂₂H₂₄ClN₃O₄ 430.15 430.0

-   10.    (2R,3R)-3-[(7-Chloro-2-ethyl-1H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 11 OH H H C₁₇H₁₆N₄O₄341.12 341.2 12 OCH₂CH₃ H H C₁₉H₂₀N₄O₄ 369.15 369.2 13 OH Cl HC₁₇H₁₅ClN₄O₄ 375.08 375.0 14 OCH₂CH₃ Cl H C₁₉H₁₉ClN₄O₄ 403.11 403.0 15OH CH₃ H C₁₈H₁₈N₄O₄ 355.13 355.2

-   11.    (2R,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid (TFA salt)-   12.    (2R,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid ethyl ester (TFA salt)-   13.    (2R,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid-   14.    (2R,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid ethyl ester-   15.    (2R,3R)-2-Hydroxy-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-4-phenyl-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 16 OH F H C₁₇H₁₅FN₄O₄359.11 359.0

-   16.    (2R,3R)-3-[(7-Fluoro-1H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 17 OH (CH₂)₃CH₃(CH₂)₃OH Cl C₂₁H₂₈ClN₃O₅ 438.17 438.2

-   17.    (2S,3R)-3-{[2-Butyl-5-chloro-3-(3-hydroxy-propyl)-3H-imidazole-4-carbonyl]-amino}-2-hydroxy-4-phenyl-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 18 OH COOH C₁₆H₁₅N₃O₆346.10 346.0 19 OH

C₂₂H₁₉N₃O₆ 422.13 422.0

-   18.    5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyrazine-2-carboxylic    acid (TFA salt)-   19.    2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyrazin-2-yl]-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 20 OH COOH C₁₇H₁₆N₂O₆345.10 345.0 21 OH

C₂₃H₂₀N₂O₆ 421.13 421.0

-   20.    5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridine-2-carboxylic    acid (TFA salt)-   21.    2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 22 OH

C₂₃H₂₀N₂O₆ 421.13 421.0 23 OCH₂CH₃

C₂₇H₂₈N₂O₆ 477.19 478.0

-   22.    2-[4-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic    acid (TFA salt)-   23.    2-[4-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic    acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 24 OH

C₂₃H₂₀N₂O₆ 421.13 421

-   24.    2-[6-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-3-yl]-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 25 OH

C₂₃H₂₀N₂O₆ 421.13 421

-   25.    2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-3-yl]-benzoic    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 26 OH

C₂₃H₂₀N₂O₆ 421.13 421.0 27 OCH₂CH₃

C₂₅H₂₄N₂O₆ 449.16 449.0 28 OCH₂CH₃

C₂₇H₂₈N₂O₆ 477.19 477.2

-   26.    2-[2-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic    acid (TFA salt)-   27.    2-[2-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic    acid (TFA salt)-   28.    2-[2-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic    acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 29 OH H C₁₆H₁₅N₅O₄ 342.11342.2

-   29.    (2R,3R)-2-Hydroxy-4-phenyl-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 30 OH C₁₈H₁₇N₃O₄ 340.12340.0

-   30.    (2R,3R)-2-Hydroxy-4-phenyl-3-[(pyrrolo[1,2-c]pyrimidine-3-carbonyl)-amino]-butyric    acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 31 OH COOH C₁₆H₁₅NO₆S350.06 350.0

-   31.    5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-thiophene-2-carboxylic    acid

Preparation 6 (R)-3-Amino-4-(2-chloro-phenyl)-butyric Acid Ethyl Ester

(R)-3-Amino-4-(2-chloro-phenyl)-butyric acid (10 g, 50 mmol) wasdissolved in absolute EtOH (250 mL). Concentrated HCl (2 mL) was addedand the mixture was heated to reflux overnight. The product was vacuumedto dryness and azeotroped with toluene (4×50 mL), then placed under highvacuum overnight to yield the title compound as a white solid (10 g).

Example 62-{2-[(R)-1-Carboxymethyl-2-(2-chloro-phenyl)-ethylcarbamoyl]-pyridin-4-yl}-benzoicAcid

4-Bromopyridine-2-carboxylic acid (159 mg, 788 μmol, 1.0 eq.), DIPEA(439 μL, 3.2 eq.) and HATU (300 mg, 788 μmol, 1.0 eq.) were combined inDCM (20 mL) and stirred for 5 minutes at room temperature.(R)-3-amino-4-(2-chloro-phenyl)-butyric acid ethyl ester (200 mg, 827μmol, 1.1 eq.) was added and the resulting mixture was stirred for hour.The reaction was quenched with saturated NH₄Cl, then extracted with DCM,dried and evaporated to yield the crude intermediate. K₂CO₃ (163 mg, 1.2mmol), 2-t-butoxycarbonylphenylboronic acid pinacol ester (264 mg, 867μmol, 1.1 eq.), EtOH (1 mL), and water (0.3 mL) were added to the crudeintermediate. The mixture was stirred, and the reaction vessel wascapped, placed under vacuum and purged with nitrogen. SilicaCat® DPP-Pd(280 μmol/g loading; 563 mg, 158 μmol) was added. The vessel wasrecapped and microwaved at 100° C. for 30 minutes. The solvent wasremoved and the product filtered. 1:1 DCM:TFA was added and theresulting mixture was stirred at 40° C. for 2 hours. The solvent wasevaporated and the crude product was dissolved in AcOH (1.5 mL) andpurified by preparative HPLC to yield the title compound as a TFA salt(50.0 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₁₉ClN₂O₅, 439.10. found 440.

Example 7

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula Ih, were prepared as TFA salts:

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 1 OH H C₁₃H₁₃ClN₄O₃309.07 309.0

-   1.    (R)-4-(2-Chloro-phenyl)-3-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 2 OH CH₃ C₁₈H₁₇ClN₄O₃373.10 373.0 3 OH H C₁₇H₁₅ClN₄O₃ 359.08 359.0 4 OH Cl C₁₇H₁₄Cl₂N₄O₃393.04 393.0

-   2.    (R)-4-(2-Chloro-phenyl)-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-butyric    acid-   3.    (R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric    acid-   4.    (R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 5 OCH₂CH₃ H C₁₉H₁₉ClN₄O₃387.11 n.d. 6 OH F C₁₇H₁₄ClFN₄O₃ 377.07 377.0 n.d. = not determined

-   5.    (R)-3-[(1H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric    acid ethyl ester-   6.    (R)-4-(2-Chloro-phenyl)-3-[(7-fluoro-1H-benzotriazole-5-carbonyl)-amino]-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 7 OH C₁₆H₁₄ClN₅O₃ 360.08360.0

-   7.    (R)-4-(2-Chloro-phenyl)-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 8 OH OH C₁₅H₁₄ClN₃O₄336.07 336.4

-   8.    (R)-4-(2-Chloro-phenyl)-3-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-butyric    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 9 OH

C₂₇H₁₈ClN₃O₃ 440.09 440.0

-   9.    2-{6-[(R)-2-Carboxy-1-(2-chloro-benzyl)-ethylcarbamoyl]-pyridazin-3-yl}-benzoic    acid

Assay 1 In Vitro Assays for the Quantitation of Inhibitor Potencies atHuman and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl) aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymeswere used at concentrations that resulted in quantitative proteolysis of1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 μM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively. Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):

v=v ₀/[1+(I/K′)]

where v is the reaction rate, v₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

Compounds of the invention were tested in this assay and found to havepK_(i) values at human NEP as follows. In general, either the prodrugcompounds did not inhibit the enzyme in this in vitro assay, or theprodrugs were not tested (n.d.) since activity would not be expected.

Ex. pK_(i) 1; R¹ = —OH; R⁴¹ = H ≧9.0 1; R¹ = —OCH₂CH₃; R⁴¹ = —CH₂CH₃n.d. 1; R¹ = —OCH₂CH₃; R⁴¹ = H n.d. 2-1 ≧9.0 2-2 ≧9.0 2-3 n.d. 2-48.0-8.9 2-5 8.0-8.9 2-6 n.d. 2-7 ≧9.0 2-8 8.0-8.9 2-9 8.0-8.9 2-108.0-8.9 2-11 8.0-8.9 2-12 8.0-8.9 2-13 8.0-8.9 2-14 8.0-8.9 2-15 8.0-8.92-16 8.0-8.9 2-17 8.0-8.9 2-18 7.0-7.9 2-19 7.0-7.9 2-20 7.0-7.9 2-217.0-7.9 2-22 7.0-7.9 2-23 7.0-7.9 2-24 8.0-8.9 2-25 n.d. 2-26 8.0-8.92-27 8.0-8.9 2-28 8.0-8.9 2-29 7.0-7.9 2-30 7.0-7.9 2-31 7.0-7.9 2-327.0-7.9 2-33 7.0-7.9 2-34 7.0-7.9 2-35 ≧9.0 2-36 7.0-7.9 2-37 8.0-8.92-38 n.d. 2-39 7.0-7.9 3 8.0-8.9 4; R¹ = —OH; R⁴¹ = H 8.0-8.9 4; R¹ =—OCH₂CH₃; R⁴¹ = H n.d. 4; R¹ = —OCH₂CH₃; R⁴¹ = —CH₂CH₃ n.d. 4; R¹ =—O(CH₂)₃CH₃; R⁴¹ =H n.d. 5-1 7.0-7.9 5-2 7.0-7.9 5-3 7.0-7.9 5-4 n.d.5-5 7.0-7.9 5-6 7.0-7.9 5-7 7.0-7.9 5-8 7.0-7.9 5-9 7.0-7.9 5-10 n.d.5-11 7.0-7.9 5-12 n.d. 5-13 7.0-7.9 5-14 n.d. 5-15 7.0-7.9 5-16 7.0-7.95-17 n.d. 5-18 7.0-7.9 5-19 7.0-7.9 5-20 7.0-7.9 5-21 8.0-8.9 5-228.0-8.9 5-23 n.d. 5-24 7.0-7.9 5-25 8.0-8.9 5-26 8.0-8.9 5-27 n.d. 5-28n.d. 5-29 7.0-7.9 5-30 7.0-7.9 5-31 8.0-8.9 6 8.0-8.9 7-1 7.0-7.9 7-27.0-7.9 7-3 7.0-7.9 7-4 7.0-7.9 7-5 n.d. 7-6 7.0-7.9 7-7 7.0-7.9 7-88.0-8.9 7-9 7.0-7.9

Pharmacodynamic (PD) Assay for ACE and NEP Activity in Anesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (flared PE 50 tubing) catheters arecannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to faciliate spontaneous respiration. The animals are then allowed a 60minute stablization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of AngI (1.0μg/kg, for ACE inhibitor activity) at 15 minutes apart. At 15 minutespost-second dose of AngI, the animals are treated with vehicle or testcompound. Five minutes later, the animals are additionally treated witha bolus i.v. injection of atrial natriuretic peptide (ANP; 30 μg/kg).Urine collection (into pre-weighted eppendorf tubes) is startedimmediately after the ANP treatment and continued for 60 minutes. At 30and 60 minutes into urine collection, the animals are re-challenged withAngI. Blood pressure measurements are done using the Notocord system(Kalamazoo, Mich.). Urine samples are frozen at −20° C. until used forthe cGMP assay. Urine cGMP concentrations are determined by EnzymeImmuno Assay using a commercial kit (Assay Designs, Ann Arbor, Mich.,Cat. No. 901-013). Urine volume is determined gravimetrically. UrinarycGMP output is calculated as the product of urine output and urine cGMPconcentration. ACE inhibition is assessed by quantifying the %inhibition of pressor response to AngI. NEP inhibition is assessed byquantifying the potentiation of ANP-induced elevation in urinary cGMPoutput.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site withfree access to food and water. For blood pressure recording, theseanimals are surgically implanted with small rodent radiotransmitters(telemetry unit; DSI Models TA 11PA-C40 or C50-PXT, Data Science Inc.,USA). The tip of the catheter connected to the transmitter is insertedinto the descending aorta above the iliac bifurcation and secured inplace with tissue adhesive. The transmitter is kept intraperitoneallyand secured to the abdominal wall while closing of the abdominalincision with a non-absorbable suture. The outer skin is closed withsuture and staples. The animals are allowed to recover with appropriatepost operative care. On the day of the experiment, the animals in theircages are placed on top of the telemetry receiver units to acclimate tothe testing environment and baseline recording. After at least of 2hours baseline measurement is taken, the animals are then dosed withvehicle or test compound and followed out to 24 hours post-dose bloodpressure measurement. Data is recorded continuously for the duration ofthe study using Notocord software (Kalamazoo, Mich.) and stored aselectronic digital signals. Parameters measured are blood pressure(systolic, diastolic and mean arterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet (8% in food or 1% NaCl in drinking water), adeoxycorticosterone acetate (DOCA) pellet (100 mg, 90 days release time,Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. At this time,the animals are also surgically implanted with small rodentradiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. Study design, data recording, and parameters measured issimilar to that described for Assay 3.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDahl/SS Rat Model of Hypertension

Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age from CharlesRiver Laboratory, USA) are allowed at least 48 hours of acclimation uponarrival at the testing site before they were placed on a 8% NaCl highsalt diet (TD.92012, Harlan, USA) then surgically implanted with smallrodent radiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. At approximately 4 to 5 weeks from the start of highsalt diet, these animals are expected to become hypertensive. Once thehypertension level is confirmed, these animals are used for the studywhile continued with the high salt diet to maintain their hypertensionlevel. Study design, data recording, and parameters measured is similarto that described in Assay 3.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1-22. (canceled) 23: A compound of: (a)(2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(2-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyricacid; (b)(2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(3-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyricacid; (c)(2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(4-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyricacid; (d)(2R,3R)-4-(2-Chloro-phenyl)-3-{[5-(2-chloro-phenyl)-isoxazole-3-carbonyl]-amino}-2-hydroxy-butyricacid; (e)(2R,3R)-3-{[2-(4-Chloro-2-fluoro-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-4-(2-chloro-phenyl)-2-hydroxy-butyricacid; (f)(2R,3R)-4-(2-Chloro-phenyl)-3-{[2-(2,3-difluoro-4-methoxy-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyricacid; (g)(2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(2-phenyl-oxazole-5-carbonyl)-amino]-butyricacid; or a pharmaceutically acceptable salt thereof. 24: Apharmaceutical composition comprising the compound of claim 23 and apharmaceutically acceptable carrier. 25: The pharmaceutical compositionof claim 24, further comprising an AT₁ receptor antagonist. 26: A methodfor treating hypertension, heart failure, or renal disease, comprisingadministering to a patient a therapeutically effective amount of thecompound of claim 23.